Process for normalizing the concentration of analytes in a urine sample

ABSTRACT

Process for normalizing the concentration of at least one analyte in a urine sample, comprising the following steps:
     a) determining the concentration of at least one analyte;   b) determining the concentration of at least one collagen or collagen fragment;   c) normalizing the concentration of said at least one analyte relative to the concentration of said at least one collagen or collagen fragment.

The present invention relates to a process for normalizing the concentration of analytes in a urine sample.

Urine samples have been used for medical laboratory examinations for a long time. Urine samples contain a variety of substances that are interesting under medical aspects, for example, degradation products of medicaments or illegal drugs, hormones, pathogens, proteins etc.

While often (only) the total protein content was recurred to for evaluating a urine sample in the past, it is recognized more and more that the composition of the proteins in urine, in particular, also contains relevant medical information.

WO 01/84140 and WO 2004/068130 describe Processes and devices for determining protein and peptide patterns in a liquid sample, for example, a urine sample.

WO 2005/024409 describes devices and processes for the quantitative evaluation of the polypeptides contained in a sample of body fluids and markers for recognizing pathological conditions. For example, it is shown how different diseases are associated with the occurrence of different polypeptide markers. It is observed that different polypeptides occur more or less frequently in certain diseases as compared to healthy patients.

WO 2006/106129 among others describes so-called amplitude markers in which two states, for example states of health and disease, are distinguished from one another, not by the occurrence, but by the amplitude, i.e., the concentration, of the markers in the sample. In order to compensate for different protein concentrations of different samples, the overall amplitude is normalized to 1 million counts after an examination of the sample by mass spectrometry in WO 2006/106129.

However, there is still a need for more specific normalization procedures for comparing different concentrations of analytes in the sample and therefore achieve a better comparability between the reference samples and measured samples.

Surprisingly, this object can be achieved by a process for normalizing the concentration of at least one analyte in a urine sample, comprising the following steps:

-   a) determining the concentration of at least one analyte; -   b) determining the concentration of at least one collagen or     collagen fragment; -   c) normalizing the concentration of said at least one analyte     relative to the concentration of said at least one collagen or     collagen fragment.

The process according to the invention has a wide variety of applications.

First, the content of analytes, collagen or collagen fragments is determined. For the determination, for example, UV/Vis detectors, fluorescence detectors, refractive index detectors, diode array detectors, mass spectrometers, gel scanners, the staining of the proteins, for example, with Coomassie brilliant blue, silver staining, Cy2, Cy3, Cy5 or the like, or the derivatization, for example, with ITRAC (isotope tags for relative and absolute qualification) and ICAT (isotope coded affinity tagging) followed by detection of the derivatized peptides may be used.

According to the invention, collagen or collagen fragments are employed for normalizing the samples. At least one collagen or collagen fragment is identified in the sample. For this purpose, identification by migration and retention times, in particular, optionally completed by mass spectra, UV/Vis spectra, fluorescence spectra or the affinity for particular ligands, for example, specific antibodies, is suitable.

The concentration of the analyte can then be normalized to the concentration of said at least one collagen or collagen fragment, i.e., the concentrations of analytes are measured relative to the concentration of the reference collagen or collagen fragments.

Preferably, however, not one collagen or collagen fragment, but several, for example, two or three or four or five or 10, 20, 30, 40 or 50 collagens or collagen fragments are employed for normalization.

In a preferred embodiment, the analyte does not comprise any hydroxypyridinium collagen cross-links, especially no hydroxylysylpyridinoline (Pyd) and deoxypyridinoline (Dpd). Preferably, none of these compounds is employed for normalizing the concentration of another analyte either.

Preferably used collagen fragments are stated in the following Table:

Seq. ID No. Protein name Swissprot name Accession number Calc. mass [M] 1 Collagen alpha-1 (I) chain [777-787] [Homo sapiens] CO1A1 Human gi115269 1016.441 2 Collagen alpha-1 (I) chain [546-558] [Homo sapiens] CO1A1 Human gi115269 1194.552 3 Collagen alpha-1 (I) chain [798-810] [Homo sapiens] CO1A1 Human gi115269 1250.553 4 Collagen alpha-1 (I) chain [546-559] [Homo sapiens] CO1A1 Human gi115269 1265.589 5 Collagen alpha-1 (III) chain [796-808] [Homo sapiens] CO3A1 Human gi115306 1268.563 6 Collagen alpha-1 (II) chain [511-524] [Homo sapiens] CO2A1 Human gi115287 1378.611 7 Collagen alpha-1 (I) chain [543-558] [Homo sapiens] CO1A1 Human gi115269 1435.658 8 Collagen alpha-1 (III) chain [642-657] [Homo sapiens] CO3A1 Human gi115306 1438.669 9 Collagen alpha-1 (I) chain [543-558] [Homo sapiens] CO1A1 Human gi115269 1451.653 10 Collagen alpha-1 (I) chain [542-558] [Homo sapiens] CO1A1 Human gi115269 1508.674 11 Collagen alpha-1 (XV) chain [1093-1109] [Homo sapiens] COFA1 Human gi68839886 1523.726 12 Collagen alpha-1 (I) chain [543-560] [Homo sapiens] CO1A1 Human gi115269 1579.711 13 Collagen alpha-1 (III) chain [587-604] [Homo sapiens] CO3A1 Human gi115306 1623.724 14 Collagen alpha-1 (I) chain [541-560] [Homo sapiens] CO1A1 Human gi115269 1737.781 15 Collagen alpha-1 (III) chain [642-661] [Homo sapiens] CO3A1 Human gi115306 1834.833 16 Collagen alpha-1 (III) chain [1174-1195] [Homo sapiens] CO3A1 Human gi115306 2025.866 17 Collagen alpha-1 (I) chain [648-669] [Homo sapiens] CO1A1 Human gi115269 2046.949 18 Collagen alpha-1 (I) chain [229-249] [Homo sapiens] CO1A1 Human gi115269 2063.926 19 Collagen alpha-1 (III) chain [586-604] [Homo sapiens] CO3A1 Human gi115306 2078.925 20 Collagen alpha-1 (I) chain [647-669] [Homo sapiens] CO1A1 Human gi115269 2087.976 21 Collagen alpha-1 (I) chain [647-669] [Homo sapiens] CO1A1 Human gi115269 2103.971 22 Collagen alpha-1 (I) chain [646-669] [Homo sapiens] CO1A1 Human gi115269 2159.013 23 Collagen alpha-1 (I) chain [432-455] [Homo sapiens] CO1A1 Human gi115269 2169.977 24 Collagen alpha-1 (I) chain [646-669] [Homo sapiens] CO1A1 Human gi115269 2175.008 25 Collagen alpha-1 (I) chain [819-843] [Homo sapiens] CO1A1 Human gi115269 2220.988 26 Collagen alpha-1 (I) chain [431-455] [Homo sapiens] CO1A1 Human gi115269 2226.999 27 Collagen alpha-1 (I) chain [699-725] [Homo sapiens] CO1A1 Human gi115269 2281.979 28 Collagen alpha-1 (I) chain [819-844] [Homo sapiens] CO1A1 Human gi115269 2292.025 29 Collagen alpha-1 (III) chain [813-840] [Homo sapiens] CO3A1 Human gi115306 2564.149 30 Collagen alpha-1 (III) chain [1168-1195] [Homo sapiens] CO3A1 Human gi115306 2679.202 31 Collagen alpha-1 (III) chain [1168-1195] [Homo sapiens] CO3A1 Human gi115306 2695.197 32 Collagen alpha-1 (III) chain [610-639] [Homo sapiens] CO3A1 Human gi115306 2742.248 33 Collagen alpha-1 (III) chain [448-477] [Homo sapiens] CO3A1 Human gi115306 2825.270 34 Collagen alpha-1 (I) chain [1011-1041] [Homo sapiens] CO1A1 Human gi115269 2942.299 35 Collagen alpha-1 (I) chain [537-569] [Homo sapiens] CO1A1 Human gi115269 3011.386 36 Collagen alpha-1 (I) chain [1011-1040] [Homo sapiens] CO1A1 Human gi115269 3013.336 37 Collagen alpha-2 (I) chain [830-865] [Homo sapiens] CO1A2 Human gi82654930 3248.533 38 Collagen alpha-1 (I) chain [1007-1041] [Homo sapiens] CO1A1 Human gi115269 3266.442 39 Collagen alpha-1 (III) chain [910-948] [Homo sapiens] CO3A1 Human gi115306 3441.603 40 Collagen alpha-1 (I) chain [630-674] [Homo sapiens] CO1A1 Human gi115269 4169.916 41 Collagen alpha-1 (III) chain [319-366] [Homo sapiens] CO3A1 Human gi115306 4289.919 42 Collagen alpha-1 (III) chain [319-366] [Homo sapiens] CO3A1 Human gi115306 4305.914

The sequences are given in FIG. 3.

The invention also relates to the use of collagen fragments for normalizing the analyte concentration of urine samples, especially with the collagen fragments as described above.

Preferred analytes whose contents are to be normalized include, in particular, proteins, peptides, lipids, metabolites, salts and other ionic compounds, carbohydrates.

The use of the process for normalizing peptide concentrations is particularly preferred.

In one embodiment, the sample is first subdivided into subsamples.

Different methods may be employed to achieve this, for example:

-   -   chromatographic methods, such as affinity chromatography,         ion-exchange chromatography, reversed-phase chromatography,         hydrophobic interaction chromatography, gel filtration,         optionally using FPLC or HPLC;     -   adsorption to specific materials, such as SELDI process,         ClinProt process;     -   electrophoretic methods, such as gel electrophoresis or         capillary electrophoresis.

The invention is further illustrated by the following Examples:

EXAMPLE 1

A urine sample was examined by means of HPLC with a downstream diode array detector (DAD). The raw date are stated in the Table. Peaks were identified by means of the retention time and spectra in the DAD. The measurement was compared with previously determined standards for these four collagen fragments, and a conversion factor was calculated for each. The average of these factors (0.80761277 in this case) was employed as a normalizing factor for all the other signals. The normalized signals are stated in the last column.

Peak Normalizing Normalized No. Signal Standard factor signal intensity 1 126.8033157 157.0100429 2 146.5503894 181.4612086 3 3115.519875 4073.54509 0.76481782 3857.690207 4 590.1256269 730.7036846 5 5461.634925 6762.690148 6 150.3823428 186.2059991 7 39.1780827 48.51097474 8 8.5271778 10.55849798 9 394.6322538 488.6404331 10 50.0342319 61.9532451 11 1577.919488 1953.807005 12 539.3141163 667.7879996 13 400.4296182 495.8188292 14 8.8306578 10.93427212 15 148.7699163 184.209465 16 538.5410307 666.8307518 17 1389.860945 1720.949688 18 93.4206021 115.6749937 19 1024.846846 1276.56782 0.80281426 1268.982963 20 2344.443075 2902.929673 21 435.1965894 538.86789 22 114.2890587 141.514675 23 15.9397308 19.73684839 24 8.9629632 11.09809494 25 398.4975981 493.4265688 26 6940.8252 8643.25876 0.80303337 8594.248947 27 76.7856591 95.07732162 28 61.2618498 75.85547438 29 64.5422931 79.9173756 30 559.1427462 692.3401497 31 323.2918818 400.3055594 32 14.1058476 17.46610272 33 399.1654701 494.2535393 34 4612.278375 5711.002284 35 174.3985782 215.9433142 36 136.4525784 168.9579257 37 909.9711288 1126.741877 38 62.1722196 76.9827099 39 119.8605303 148.4133668 40 2922.828975 3399.48571 0.85978563 3619.097026 41 2.625588 3.251048147 42 52.8236532 65.40715445 43 178.5294621 221.0582456 0.80761277

EXAMPLE 2

A urine sample was separated by means of 2D electrophoresis as described in Thongboonkerd et al. in Kidney Int 62: 1461-1469 (2002), over a pH range of from 3 to 10 and using a 10% polyacrylamide gel having a length of 22 cm. The gel was stained with Coomassie stain, and the collagens were detected in a gel prepared in parallel by means of immunological methods (Western blot). Predetermined standards for these five proteins were employed, and a linear regression performed. With this calibration formula, the further values were converted. The formula was Y=103.82x−127449. The R² value was 0.957.

Peak No. Signal Standard Normalized signal intensity 1 4508518.988 467946992.3 2 153935103 15981414944 3 23582568 2448214761 4 44251661.9 4594080090 5 586248.746 60736895.81 6 948640.866 98360445.71 7 1383514.774 143509054.8 8 122812941 12750312086 9 2944789.572 305600604.4 10 30550952.65 3171672456 11 1089289.56 117284244.8 112962593.1 12 577112.586 59788379.68 13 1130932.024 117285913.7 14 913664.024 94729149.97 15 397773.048 41169348.84 16 6931703.714 719522030.6 17 38342270.25 3980567048 18 2391089.672 248115480.7 19 2250316.422 233500401.9 20 932726.014 96708165.77 21 10695075.82 1110235323 22 1438795.038 149248251.8 23 8548926.298 887422079.3 24 7982762.024 828642904.3 25 519540.4375 58011688.21 53811239.22 26 4877082.25 506211230.2 27 27564764.65 2861646417 28 20622151.95 2140864367 29 3862566.202 400884174.1 30 219714.4865 23759647.58 22683308.99 31 13240495.55 1374500799 32 6102716.5 633456578 33 3523566.41 365689215.7 34 1011312.708 104867036.3 35 10681983.25 1108876052 36 390792.574 40444636.03 37 3552944.744 368739274.3 38 8037397.154 834315123.5 39 1966378.988 204022017.5 40 89404658.25 9281864171 41 2051887.75 212899537.2 42 7424138.214 770646580.4 43 32377626.4 3361317724 44 34631829 3595349038 45 1948609.006 202177138 46 107055624.3 11114387461 47 10008414.75 1038946170 48 3575878.756 371120283.4 49 1359764.006 141043250.1 50 631532.685 52349499.17 65438274.36 51 1981878.154 205631140.9 52 6658241.834 691131218.2 53 8132593.25 844198382.2 54 5853332.798 607565562.1 55 10995087.2 1141382504 56 3309612.762 343476548 57 5692416.786 590859261.7 58 3166113.976 328578504 59 20333775.95 2110925170 60 32900793.65 3415632948 61 5041336.916 523264149.6 62 2926167.976 303667310.3 63 10445490.05 1084323328 64 775850.282 80421327.28 65 339214.044 35089753.05 66 41363211.15 4294201133 67 23456220.45 2435097358 68 15788615 1639046560 69 34321605.15 3563141598 70 897196.084 93019448.44 71 327241.543 37343483.31 33846767.99 72 9414104.178 977244846.8 73 1965153.274 203894763.9 74 45057999.65 4677794075 75 877939.968 91020278.48 76 3120269.268 323818906.4 77 3584994.964 372066728.2 78 10224568.98 1061387302 79 735893.154 76272978.25 80 5907069.334 613144489.3 81 36156493.15 3753639670 82 10877155.98 1129138884 83 8374707.928 869334728.1 84 16689498.2 1732576254 85 5895062.464 611897936 86 1706771.568 177069575.2

The quality of the regression is shown in FIG. 1.

EXAMPLE 3

A urine sample was examined by capillary electrophoresis coupled with mass spectrometry. Twenty-seven collagen fragments could be identified thereby, which were then compared with previously determined standards. This was followed by normalization by means of linear regression with the proviso that the straight line must go through the value zero. Corresponding measuring data and the calibration are shown in the following Table.

No. CE time (min) Mass Signal Standard Normalized signal intensity 1 19.948 17266.043 184882.922 1368.13362 2 19.944 17119.789 587371.188 4346.54679 3 19.931 17062.977 240560 1780.144 4 19.946 16972.203 352392.875 2607.70728 5 19.908 16952.465 197797.906 1463.7045 6 19.931 16918.75 2107725.75 15597.1706 7 19.873 16806.326 124057.313 918.024116 8 19.917 16771.715 1365796.63 10106.895 9 22.387 10342.543 119401.375 883.570175 10 20.699 9905.148 89822.563 664.686966 11 20.727 9867.076 526946.5 3899.4041 12 20.599 9626.376 176806.484 1308.36798 13 20.904 8875.947 124966.711 924.753661 14 20.927 8837.85 796843.125 5896.63913 15 19.926 8578.571 17265.779 127.766765 16 20.156 8289.725 286028.781 2116.61298 17 20.078 8176.791 674227.938 4989.28674 18 57.332 6788.82 438.533 3.2451442 19 19.251 6491.179 95395.688 705.928091 20 23.865 6329.103 62460.586 462.208336 21 19.542 6308.096 23674.088 175.188251 22 20.842 6237.104 134147.797 992.693698 23 23.72 6225.298 67887.742 502.369291 24 23.66 6208.243 85896.305 635.632657 25 23.741 6186.583 288461.313 2134.61372 26 23.643 6170.012 399146.313 2953.68272 27 19.662 5861.38 218662.969 1618.10597 28 22.258 5635.462 14248.398 105.438145 29 22.266 5612.497 112633.008 833.484259 30 22.301 5574.535 988766.188 7316.86979 31 20.377 5118.564 18909.668 139.931543 32 20.441 5096.631 93874.516 694.671418 33 20.448 5058.56 250174.453 1851.29095 34 23.737 5039.495 283460.031 2097.60423 35 22.982 5038.431 174797.016 1293.49792 36 20.431 5036.265 628288.625 4649.33583 37 23.001 5023.362 43507.188 321.953191 38 20.438 5020.505 641471.938 4746.89234 39 23.009 5000.172 753943.563 5579.18237 40 20.45 4998.592 2524795 18683.483 41 20.409 4982.02 313462.719 2319.62412 42 20.549 4976.683 1167032.75 8636.04235 43 52.301 4964.782 358.613 2.6537362 44 20.466 4960.742 14400323 106562.39 45 20.358 4933.646 23247.875 1652.03428 46 22.651 4876.404 51850.109 383.690807 47 38.197 4861.141 494.304 3.6578496 48 22.682 4838.021 281694.156 2084.53675 49 22.655 4822.251 52409.906 387.833304 50 22.683 4800.063 1058541.25 7833.20525 51 48.573 4793.838 282.485 2.090389 52 55.222 4777.634 374.187 2.7689838 53 39.763 4774.006 532.92 3.943608 54 48.826 4772.17 272.009 2.0128666 55 57.032 4766.609 339.383 2.5114342 56 19.62 4730.656 24256.555 179.498507 57 37.899 4719.636 2192.44 16.224056 58 23.902 4692.252 68363.453 505.889552 59 54.464 4690.256 358.075 2.649755 60 22.332 4671.967 96398.766 713.350868 61 25.889 4668.661 32946.504 243.80413 62 21.078 4656.171 37012.164 273.890014 63 23.915 4654.204 252635.781 1869.50478 64 19.366 4651.778 422.893 3.1294082 65 25.936 4630.038 76921.227 569.21708 66 54.567 4620.673 895.427 6.6261598 67 23.396 4619.578 16829.051 124.534977 68 52.701 4602.985 298.8 2.21112 69 58.14 4602.554 423.734 3.1356316 70 57.637 4594.426 391.032 2.8936368 71 52.603 4577.292 340.97 2.523178 72 20.741 4566.496 232446.344 1720.10295 73 49.024 4560.303 642.982 4.7580668 74 54.011 4559.723 4928.111 36.4680214 75 18.86 4552.372 345.112 2.5538288 76 51.846 4548.482 373.052 2.7605848 77 26.066 4547.384 12106.5 89.5881 78 51.365 4544.345 1851.577 13.7016698 79 53.986 4543.723 22582.416 167.109878 80 54.024 4520.931 4122.547 30.5068478 81 40.87 4520.884 552.198 4.0862652 82 25.763 4468.206 33454.656 247.564454 83 25.839 4452.583 21824.857 161.503942 84 48.976 4445.646 571.726 4.2307724 85 20.501 4443.026 18179.678 134.529617 86 27.642 4439.813 97056.883 718.220934 87 24.137 4436.181 404617.531 2994.16973 88 58.694 4420.792 432.338 3.1993012 89 25.813 4414.273 15183.697 112.359358 90 19.965 4410.955 338675.063 2506.19547 91 20.515 4405.046 291842.344 2159.63335 92 25.558 4404.052 42032.641 311.041543 93 20.055 4391.25 224259.906 1659.5233 94 59.551 4390.04 363.251 2.6880574 95 59.07 4383.307 677.349 5.0123826 96 20.018 4375.046 105928.648 783.871995 97 25.571 4366.142 216682.813 1603.45282 98 20.066 4353.102 1819283.25 13462.6961 99 22.383 4349.45 216404.641 1601.39434 100 25.569 4344.106 288232.313 2132.91912 101 20.337 4334.652 45377.477 335.79333 102 25.603 4328.157 542624 4015.4176 103 60.458 4319.592 330.163 2.4432062 104 27.298 4307.342 30545.982 226.040267 105 25.614 4306.212 529838.313 3100.93516 3920.80352 106 18.866 4302.308 277.252 2.0516648 107 25.618 4290.197 708976.625 3872.09443 5246.42703 108 25.58 4274.179 57546.574 425.844648 109 25.607 4252.228 98721.375 730.538175 110 23.903 4239.119 31304.807 231.655572 111 20.202 4233.57 31109.875 230.213075 112 25.741 4229.222 27980.471 207.055485 113 23.93 4217.721 698545.563 5169.23717 114 28.392 4208.093 85557.367 633.124516 115 20.629 4196.917 227998.453 1687.18855 116 28.393 4192.087 152065.688 1125.28609 117 27.019 4180.957 29489.236 218.220346 118 28.396 4170.166 176045.016 1291.45231 1302.73312 119 23.8 4160.604 154588.094 1143.9519 120 22.532 4160.021 318391.75 2356.09895 121 27.033 4160.011 69551.938 514.684341 122 28.349 4154.089 35176.504 260.30613 123 27.009 4144.035 62706.73 464.029802 124 22.513 4144.034 67626.914 500.439164 125 23.294 4135.927 40501.199 299.708873 126 22.526 4122.078 1111014.88 8221.51008 127 27.052 4121.97 143200.75 1059.68555 128 26.993 4106.017 56078.742 414.982691 129 20.242 4105.763 27811.58 205.805692 130 28.055 4102.563 18823.371 139.292945 131 27.022 4084.008 103050.93 762.576882 132 28.057 4082.969 35312.172 261.310073 133 28.103 4063.103 70996.594 525.374796 134 20.494 4062.357 192299.219 1423.01422 135 20.46 4046.127 45463.988 336.433511 136 20.849 4044.743 40488.367 299.613916 137 28.08 4044.394 166345.453 1230.95635 138 20.276 4044.247 575594.75 4259.40115 139 20.865 4028.943 54097.566 400.321988 140 20.601 4025.685 273715.438 2025.49424 141 28.152 4025.156 75278.781 557.062979 142 25.055 4015.3 13494.786 99.8614164 143 20.423 4009.552 46446.355 343.703027 144 20.88 4007.199 241439.844 1786.65485 145 28.076 4006.013 98433.141 728.405243 146 20.719 3996.902 309862.719 2292.98412 147 20.438 3986.617 1697006.63 12557.849 148 20.881 3968.808 427206.25 3161.32625 149 19.8 3960.04 129275.078 956.635577 150 19.704 3913.814 25934.791 191.917453 151 19.645 3900.239 29670.904 219.56469 152 19.631 3877.959 85786.75 634.82195 153 19.619 3861.996 59334.113 439.072436 154 23.401 3860.224 31707.752 234.637365 155 19.105 3856.541 110644.117 818.766466 156 28.332 3839.987 28982.951 214.473837 157 19.659 3839.739 105607.102 781.492555 158 25.36 3832.015 15375.597 113.779418 159 23.419 3822.288 21333.066 157.864688 160 20.048 3814.276 379764.75 2810.25915 161 28.373 3801.977 68342.133 505.731784 162 27.677 3801.896 13939.659 103.153477 163 27.719 3794.861 51831.453 383.552752 164 21.453 3782.819 17585.135 130.129999 165 31.428 3775.772 53642.129 396.951755 166 27.744 3772.916 129047.891 954.954393 167 21.446 3760.889 121781.781 901.185179 168 19.224 3760.068 42083.434 311.417412 169 21.836 3757.834 12074.36 89.350264 170 27.7 3756.982 327179.375 2421.12738 171 21.886 3741.82 31431.242 232.591191 172 27.715 3739.881 56333.398 416.867145 173 27.678 3735.445 121638.703 900.126402 174 19.018 3729.583 19175.627 141.89964 175 21.454 3722.747 1143063.38 8458.66898 176 21.905 3720.201 182391.875 1349.69988 177 27.69 3718.93 211491.453 1565.03675 178 19.466 3710.955 28338.004 209.70123 179 21.427 3706.46 187850.703 1390.0952 180 21.882 3703.562 156398.141 1157.34624 181 27.706 3702.282 83424.43 617.340782 182 27.527 3699.89 27447.908 203.114519 183 33.259 3695.777 61599.129 455.833555 184 21.69 3686.37 179506.266 1328.34637 185 31.253 3674.799 30919.297 228.802798 186 22.842 3669.826 17117.689 126.670899 187 18.862 3662.674 745.133 5.5139842 188 33.247 3657.754 144639.984 1070.33588 189 21.345 3630.767 245992.188 1820.34219 190 19.415 3620.771 139033.516 1028.84802 191 28.076 3616.437 45247.488 334.831411 192 19.346 3605.381 42433.102 314.004955 193 26.967 3593.676 72137.609 533.818307 194 19.373 3582.885 557225.625 4123.46963 195 27.002 3571.638 158852.625 1175.50943 196 27.423 3562.87 16845.193 124.654428 197 23.372 3559.398 131749.125 974.943525 198 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152.059218 612 28.626 2396.215 186626 1381.0324 613 54.355 2395.78 17539.246 129.79042 614 19.791 2395.33 38127.117 282.140666 615 53.832 2395.108 25210.896 186.56063 616 22.181 2393.58 28325.973 209.6122 617 37.389 2392.069 32789.227 242.64028 618 36.842 2392.022 16480.713 121.957276 619 53.509 2390.96 7808.705 57.784417 620 55.017 2390.746 56777.559 420.153937 621 21.858 2389.367 1415681.5 10476.0431 622 28.783 2384.878 67750.172 501.351273 623 54.213 2381.804 9437.86 69.840164 624 53.96 2379.819 13638.621 100.925795 625 28.586 2379.768 179069.688 1325.11569 626 20.738 2377.238 713037.375 5276.47658 627 28.792 2377.105 243979.281 1805.44668 628 54.034 2374.369 98473.961 728.707311 629 38.119 2370.071 587311.313 4346.10372 630 55.054 2368.829 45047.953 333.354852 631 49.833 2368.79 7574.214 56.0491836 632 21.712 2364.035 14890.036 110.186266 633 47.251 2363.877 169502.969 1254.32197 634 47.112 2359.46 226706.625 1677.62903 635 54.06 2358.796 271180 2006.732 636 28.638 2358.069 484792 3587.4608 637 30.392 2356.841 24718.348 182.915775 638 49.349 2356.439 49754.129 368.180555 639 22.229 2355.161 64185.695 474.974143 640 37.22 2354.086 71290.633 527.550684 641 54.995 2351.738 14965.655 110.745847 642 19.26 2343.309 31234.203 231.133102 643 54.345 2342.704 55254.418 408.882693 644 21.753 2342.053 57971.406 428.988404 645 53.764 2341.883 102171.742 756.070891 646 28.619 2341.757 395942.094 2929.9715 647 24.143 2341.072 185665.328 1373.92343 648 28.832 2339.135 119528.75 884.51275 649 54.092 2336.821 849316.625 6284.94303 650 49.582 2334.924 16711.031 123.661629 651 49.932 2332.825 7154.382 52.9424268 652 38.111 2332.118 724905.563 5364.30117 653 28.429 2332.011 26272.477 194.41633 654 49.784 2330.848 18374.775 135.973335 655 20.777 2329.976 13845.191 102.454413 656 20.781 2328.12 114579.438 847.887841 657 21.695 2326.051 33955.465 251.270441 658 28.514 2325.829 27821.584 205.879722 659 24.109 2325.039 347883.406 2574.3372 660 18.808 2323.646 3501.718 25.9127132 661 21.857 2322.105 27377.75 202.59535 662 54.036 2320.85 1445477 10696.5298 663 21.736 2320.083 17368.453 128.526552 664 28.756 2320.083 1394689.88 10320.7051 665 31.151 2319.995 18706.303 138.426642 666 50.101 2319.65 21160.838 156.590201 667 49.596 2318.781 65589.969 485.365771 668 53.96 2314.817 5407.608 40.0162992 669 28.434 2313.129 21076.289 155.964539 670 20.695 2312.394 183844.266 1360.44757 671 45.447 2310.83 8350.151 61.7911174 672 28.377 2309.901 32903.531 243.486129 673 24.091 2309.052 53750.734 397.755432 674 28.352 2308.815 65167.617 482.240366 675 19.413 2306.093 11910.861 88.1403714 676 47.374 2305.43 20267.41 149.978834 677 30.479 2304.938 64574.715 477.852891 678 54.005 2304.69 601818.875 4453.45968 679 55.551 2304.254 20926.719 154.857721 680 28.66 2304.2 441698.625 3268.56983 681 21.748 2304.056 491680.813 3638.43802 682 54.026 2298.86 2803671 20747.1654 683 20.539 2298.221 189552.234 1402.68653 684 30.798 2295.988 10958.861 81.0955714 685 50.105 2295.884 10295.519 76.1868406 686 19.179 2295.451 24928.195 184.468643 687 28.816 2295.237 260138.016 1925.02132 688 28.385 2295.094 55681.145 412.040473 689 21.739 2288.101 57605.777 426.28275 690 24.096 2287.057 1262872.75 9345.25835 691 28.388 2287.048 297568.75 2202.00875 692 20.5 2286.111 21349.635 157.987299 693 24.027 2283.095 24350.102 180.190755 694 54.068 2282.898 2728088.75 20187.8568 695 28.732 2282.105 667956.938 6281.39651 4942.88134 696 21.782 2282.103 58645.039 433.973289 697 30.387 2282 115145.461 852.076411 698 38.199 2281.901 31642.785 234.156609 699 38.703 2281.894 8948.516 66.2190184 700 40.729 2281.884 69357.18 513.243132 701 37.847 2281.877 7632.981 56.4840594 702 49.628 2280.479 11230.125 83.102925 703 53.207 2278.702 26455.098 195.767725 704 28.903 2273.115 293589.719 2172.56392 705 28.157 2272.659 27751.025 205.357585 706 28.368 2271.091 240296.203 1778.1919 707 23.951 2271.069 46381.387 343.222264 708 54.563 2266.722 53080.328 392.794427 709 53.746 2266.569 194273.734 1437.62563 710 21.735 2266.113 834853.813 6177.91822 711 47.142 2264.341 11589.805 85.764557 712 25.004 2264.038 10342.722 76.5361428 713 54.111 2260.906 3409623 25231.2102 714 39.005 2259.881 8171.715 60.470691 715 38.018 2259.88 89646.383 663.383234 716 29.265 2257.021 369411.719 2733.64672 717 28.144 2250.717 73907.656 546.916654 718 28.424 2249.076 762669.688 5643.75569 719 20.437 2249.042 143049.578 1058.56688 720 47.446 2248.591 114672.375 848.575575 721 53.175 2246.379 18363.697 135.891358 722 54.105 2244.921 1221368.75 9038.12875 723 53.209 2240.749 57652.742 426.630291 724 49.832 2237.759 5953.025 44.052385 725 28.924 2235.175 214885.766 1590.15467 726 28.13 2234.935 156541.781 1158.40918 727 28.391 2233.099 305448.406 2260.3182 728 20.474 2232.684 41022.949 303.569823 729 25.029 2231.969 11965.051 88.5413774 730 23.853 2230.051 38128.531 282.151129 731 25.127 2227.077 31312.469 231.712271 732 47.253 2226.358 138834.063 1027.37207 733 24.982 2226.04 37025.727 273.99038 734 54.067 2222.955 1451186.5 10738.7801 735 55.587 2222.909 18411.467 136.244856 736 39.072 2221.946 758603.125 5613.66313 737 33.365 2221.747 13675.914 101.201764 738 49.924 2221.487 22766.725 168.473765 739 28.601 2216.12 62841.969 465.030571 740 21.813 2215.931 38498.379 284.888005 741 28.165 2212.947 407865.344 3018.20355 742 20.541 2211.332 175885.453 1301.55235 743 28.425 2211.08 604871.438 4476.04864 744 47.358 2210.38 198223.844 1466.85645 745 24.99 2209.092 148361.516 1097.87522 746 27.351 2208.986 43820.238 324.269761 747 34.776 2208.878 7891.04 58.393696 748 23.983 2208.045 1445503.75 10696.7278 749 33.037 2207.824 20242.492 149.794441 750 53.207 2207.742 5340.262 39.5179388 751 29.961 2206.693 69426.469 513.755871 752 39.013 2205.963 658740.688 4874.68109 753 54.008 2203.897 11335.302 83.8812348 754 53.189 2202.779 65413.605 484.060677 755 19.293 2200.036 22179.658 164.129469 756 55.068 2199.737 7793.02 57.668348 757 28.127 2197.011 487901.156 3610.46855 758 20.181 2194.916 131013.984 969.503482 759 50.327 2194.728 41764.328 309.056027 760 24.981 2193.839 73573.773 544.44592 761 20.446 2193.108 20270.781 150.003779 762 54.947 2192.113 67683.648 500.858995 763 23.925 2192.015 526681.563 3897.44357 764 21.89 2191.903 167538 1239.7812 765 50.864 2191.723 32037.535 237.077759 766 47.244 2188.395 251956.906 1864.4811 767 32.649 2188.058 78885.688 583.754091 768 24.014 2186.089 116699.281 863.574679 769 29.978 2184.703 233078.734 1724.78263 770 39.09 2183.992 1648277.63 12197.2544 771 50.193 2178.759 33248.797 246.041098 772 54.895 2176.709 42103.77 311.567898 773 21.423 2175.984 37351.434 276.400612 774 51.031 2175.813 37432.242 276.998591 775 28.214 2175.135 523507.813 4308.36938 3873.95782 776 55.139 2174.695 12196.703 90.2556022 777 47.254 2172.427 139909.953 1035.33365 778 24.955 2171.845 392253.938 2902.67914 779 54.95 2170.835 83249.852 616.048905 780 23.99 2170.06 2801978.75 16950.086 20734.6428 781 33.051 2169.816 115270.117 852.998866 782 28.029 2169.083 41560.289 307.546139 783 29.942 2168.741 50702.242 375.196591 784 38.918 2168 625043.188 4625.31959 785 47.105 2167.452 166361.109 1231.07221 786 23.628 2166.176 62101.336 459.549886 787 27.877 2163.994 17963.393 132.929108 788 19.292 2163.03 26186.1 193.77714 789 28.177 2159.127 165196.703 2003.94316 1222.4556 790 21.729 2157.014 371334.938 2747.87854 791 50.303 2156.445 98210.313 726.756316 792 24.927 2156.036 134981.219 998.861021 793 54.905 2155.728 215793.328 1596.87063 794 50.291 2153.919 356736.531 2639.85033 795 33.038 2153.853 17994.725 133.160965 796 24.073 2153.761 687819.375 5089.86338 797 21.431 2151.975 13115.051 97.0513774 798 47.014 2150.457 80450.156 595.331154 799 24.985 2150.074 124303.188 919.843591 800 23.697 2148.078 14427.769 106.765491 801 47.017 2146.51 33126.008 245.132459 802 39.031 2146.033 1517131 11226.7694 803 28.033 2142.049 116757.086 864.002436 804 50.317 2140.053 49425.555 365.749107 805 54.907 2139.687 131492.109 973.041607 806 19.34 2139.264 16454.809 121.765587 807 21.438 2138.028 170402.016 1260.97492 808 49.775 2137.904 122028.063 903.007666 809 51.01 2137.833 107543.789 795.824039 810 24.98 2134.064 184060.313 1362.04632 811 23.947 2133.997 14915.034 110.371252 812 54.93 2133.748 448603.344 3319.66475 813 23.791 2132.926 98156.297 726.356598 814 33.034 2131.834 93856.258 694.536309 815 49.73 2131.826 40604.926 300.476452 816 19.309 2126.895 11998.439 88.7884486 817 19.23 2125.959 13855.418 102.530093 818 28.013 2125.696 179600.594 1329.0444 819 29.033 2123.978 9567.942 70.8027708 820 28.424 2123.872 38521.063 285.055866 821 19.288 2123.304 41390.043 306.286318 822 54.923 2117.754 676757.125 5008.00273 823 50.182 2115.807 562227.938 4160.48674 824 32.559 2113.632 14000.153 103.601132 825 27.977 2110.026 15948.838 118.021401 826 19.242 2109.934 16406.662 121.409299 827 23.748 2108.995 14636.829 108.312535 828 27.756 2106.99 38564.801 285.379527 829 20.492 2105.879 76712.156 567.669954 830 54.924 2101.744 308647.25 2283.98965 831 21.456 2101.398 246869.797 1826.8365 832 49.813 2099.871 64825.379 479.707805 833 50.969 2099.86 21705.629 160.621655 834 22.512 2098.949 30868.66 228.428084 835 54.919 2095.774 1133086.88 8384.84288 836 56.91 2094.771 16988.391 125.714093 837 50.601 2094.588 22926.068 169.652903 838 23.784 2093.206 75977.586 562.234136 839 28.084 2088.017 171200.609 1269.99462 1266.88451 840 19.264 2087.973 350660.688 2594.88909 841 23.117 2086.65 12786.285 94.618509 842 27.791 2085.045 199885.375 1479.15178 843 20.269 2081.018 229086.047 1695.23675 844 54.93 2079.801 993210.188 7349.75539 845 47.595 2079.318 19015.662 140.715899 846 56.839 2078.89 19401.766 143.573068 847 49.785 2077.893 115355.531 853.630929 848 21.811 2077.762 31063.748 229.871735 849 52.075 2077.73 6613.918 48.9429932 850 21.41 2076.984 175348.891 1297.58179 851 51.543 2071.648 7766.515 57.472211 852 23.759 2071.002 133591.328 988.575827 853 21.424 2070.003 109701.07 811.787918 854 51.254 2069.625 61727.754 456.78538 855 27.817 2069.09 222562.547 1646.96285 856 20.532 2067.921 477863.719 3536.19152 857 27.607 2063.948 71420.422 528.511123 858 54.94 2063.804 166864.938 1234.80054 859 55.581 2062.783 10409.057 77.0270218 860 22.44 2059.045 31675.367 234.397716 861 54.9 2057.817 1045255.63 7734.89163 862 20.802 2056.804 23316.27 172.540398 863 47.411 2056.32 114767.031 849.276029 864 23.834 2056.014 75674.141 559.988643 865 52.099 2054.689 8212.415 60.771871 866 51.141 2052.646 7170.431 53.0611894 867 23.596 2050.953 30659.434 226.879812 868 23.173 2049.013 179035.266 1324.86097 869 51.154 2047.635 93990.273 695.52802 870 27.801 2047.175 411581 1703.37702 3045.6994 871 28.43 2045.938 76368.227 565.12488 872 28.078 2041.99 11063.896 81.8728304 873 54.923 2041.771 361323.031 2673.79043 874 21.426 2039.213 401444.531 2970.68953 875 40.128 2036.857 62552.297 462.886998 876 40.967 2036.734 16498.773 122.09092 877 19.377 2035.142 119280.563 882.676166 878 20.833 2034.878 107200.859 793.286357 879 51.167 2031.663 179833.078 1330.76478 880 27.821 2031.125 226310.281 1674.69608 881 49.933 2030.735 8329.54 61.638596 882 20.301 2029.874 59304.598 438.854025 883 27.841 2027.979 24592.67 181.985758 884 27.615 2025.995 173888.672 1286.77617 885 21.281 2023.988 15667.275 115.937835 886 54.897 2019.862 339981.563 2515.86357 887 47.368 2018.356 144909.141 1072.32764 888 20.769 2017.852 41804.027 309.3498 889 50.41 2017.309 23882.344 176.729346 890 52.145 2016.741 25066.926 185.495252 891 51.168 2015.674 15682.338 116.049301 892 21.462 2014.872 676609.125 5006.90753 893 23.598 2013.999 118936.234 880.128132 894 27.425 2013.957 10481.886 77.5659564 895 27.626 2009.933 150281.469 1112.08287 896 51.219 2009.681 269662.813 1995.50482 897 51.765 2008.693 8050.752 59.5755648 898 27.027 2008.114 36968.852 273.569505 899 49.949 2007.869 263181.844 1947.54565 900 53.968 2007.765 23462.338 173.621301 901 47.566 2003.37 5718.014 42.3133036 902 20.834 1996.814 335825.125 2485.10593 903 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20.19 1942.355 176231.625 1304.11403 953 36.009 1939.849 754929.75 5586.48015 954 21.083 1939.728 83881.531 620.723329 955 27.622 1937.032 122677.93 907.816682 956 20.174 1935.815 14819.995 109.667963 957 55.67 1935.509 1866.308 13.8106792 958 19.929 1934.88 185292.125 1371.16173 959 27.6 1933.946 21970.883 162.584534 960 21.312 1933.467 120517.273 891.82782 961 23.44 1933.132 1264035.38 9353.86178 962 49.882 1931.835 169525.344 1254.48755 963 53.987 1931.539 7912.462 58.5522188 964 32.897 1930.835 60372.492 446.756441 965 40.453 1929.642 1351748.75 10002.9408 966 49.985 1928.736 30871.664 228.450314 967 19.413 1928.014 24680.447 182.635308 968 22.284 1925.639 28804.176 213.150902 969 36.02 1923.804 788593 5835.5882 970 32.585 1923.744 18391.229 136.095095 971 51.474 1923.542 22426.631 165.957069 972 21.155 1921.892 25471.947 188.492408 973 20.197 1920.513 484588.406 3585.9542 974 35.145 1917.819 151947.547 1124.41185 975 20.275 1916.865 592958 4387.8892 976 32.944 1914.85 53011.285 392.283509 977 39.532 1913.055 359803.531 2662.54613 978 27.873 1912.956 171374.188 1268.16899 979 49.886 1912.77 90604.273 670.47162 980 23.536 1911.131 16659075 123277.155 981 19.165 1909.169 16568.082 122.603807 982 35.112 1907.913 132959.188 983.897991 983 51.45 1907.405 55725.84 412.371216 984 20.208 1904.837 417636.094 3090.5071 985 19.974 1901.937 12937.851 95.7400974 986 36.035 1901.931 1493067.88 11048.7023 987 27.44 1901.067 35484.109 262.582407 988 21.174 1898.892 49621.984 367.202682 989 26.836 1897.634 51880.426 383.915152 990 22.569 1895.801 31370.744 232.143506 991 50.839 1895.655 14071.792 104.131261 992 19.364 1893.904 10081.896 74.6060304 993 20.096 1893.708 16126.631 119.337069 994 23.44 1893.144 138012.906 1021.2955 995 33.628 1892.875 666429.375 4931.57738 996 40.178 1891.728 965003.063 7141.02267 997 23.747 1890.939 199265.828 1474.56713 998 28.18 1889.913 10303.565 76.246381 999 51.81 1886.097 32181.391 238.142293 1000 49.855 1886.017 9415.063 69.6714662 1001 19.928 1885.96 15694.74 116.141076 1002 35.93 1885.943 758995.75 5616.56855 1003 51.337 1885.571 114489.086 847.219236 1004 20.18 1882.925 4423241 32731.9834 1005 29.37 1882.543 68798.625 509.109825 1006 35.135 1879.95 58723.824 434.556298 1007 22.494 1879.257 20705.602 153.221455 1008 34.349 1878.875 33668.203 249.144702 1009 22.591 1876.914 37062.238 274.260561 1010 21.725 1876.902 297599.3131 1550.63508 2202.23492 1011 32.986 1876.876 128455.727 950.57238 1012 55.924 1875.624 2100.854 15.5463196 1013 26.625 1875.501 105028.836 777.213386 1014 27.296 1874.767 67852.57 502.109018 1015 26.891 1873.891 93489.258 691.820509 1016 21.431 1872.932 23245.711 172.018261 1017 19.83 1872.86 146425.875 1083.55148 1018 51.282 1869.592 141043.234 1043.71993 1019 20.63 1867.838 31031.188 229.630791 1020 27.992 1864.675 33520.336 248.050486 1021 47.391 1864.278 57918.516 428.597018 1022 20.099 1864.101 137271.891 1015.81199 1023 35.869 1863.967 524659.438 3882.47984 1024 27.239 1863.873 52788.363 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1380.342 13301.506 98.4311444 1717 25.679 1378.704 342898.781 2415.39444 2537.45098 1718 49.632 1378.394 5379.274 39.8066276 1719 18.97 1377.85 30348.645 224.579973 1720 25.346 1375.624 46687.473 345.4873 1721 32.125 1375.532 67528.18 499.708532 1722 37.369 1375.445 8201.093 60.6880882 1723 34.801 1375.438 17825.877 131.91149 1724 55.31 1374.42 31385.559 232.253137 1725 56.785 1374.415 9927.623 73.4644102 1726 32.405 1373.689 38635.266 285.900968 1727 60.227 1373.36 6000.033 44.4002442 1728 45.05 1368.949 34093.426 252.291352 1729 32.464 1367.728 132403.016 979.782318 1730 55.22 1367.496 1103.651 8.1670174 1731 60.375 1366.465 12431.157 91.9905618 1732 25.359 1365.647 47731.473 353.2129 1733 45.052 1365.037 104198.375 771.067975 1734 19.189 1364.748 11513.005 85.196237 1735 25.813 1364.605 75452.961 558.351911 1736 25.024 1364.429 298666.156 2210.12955 1737 51.066 1363.59 4498.999 33.2925926 1738 30.979 1363.458 95695.992 708.150341 1739 45.02 1362.89 110947.891 821.014393 1740 32.068 1359.662 52750.859 390.356357 1741 25.534 1359.656 1906445.75 14107.6986 1742 31.049 1358.466 151038.281 1117.68328 1743 49.608 1356.381 45604.996 337.47697 1744 59.708 1356.319 29863.813 220.992216 1745 60.659 1355.61 1210.575 8.958255 1746 55.572 1354.558 1049.276 7.7646424 1747 58.894 1354.391 2738.662 20.2660988 1748 23.999 1353.72 101441.016 750.663518 1749 23.416 1352.847 43856.227 324.53608 1750 30.605 1352.398 90038.555 666.285307 1751 32.39 1351.686 32230.543 238.506018 1752 34.799 1351.387 8059.731 59.6420094 1753 25.989 1350.605 40075.652 296.559825 1754 59.605 1350.549 6613.854 48.9425196 1755 59.372 1349.363 5556.154 41.1155396 1756 25.073 1348.535 290868.375 2152.42598 1757 18.811 1347.718 12626.824 93.4384976 1758 25.458 1343.664 766104.375 5669.17238 1759 26.465 1342.628 17343.17 128.339458 1760 53.156 1342.392 5754.031 42.5798294 1761 45.041 1341.946 191931.266 1420.29137 1762 29.936 1339.384 45313.438 335.319441 1763 22.748 1338.66 77636.578 574.510677 1764 60.353 1338.395 2928.564 21.6713736 1765 30.621 1336.417 37646.219 278.582021 1766 55.091 1334.981 17385.801 128.654927 1767 59.445 1334.35 36656.449 271.257723 1768 19.066 1332.751 11472.639 84.8975286 1769 25.099 1332.551 113287.906 838.330504 1770 59.549 1330.309 2890.745 21.391513 1771 59.275 1330.277 9237.706 68.3590244 1772 25.154 1326.551 949441.625 7025.86803 1773 45.045 1324.926 42727.93 316.186682 1774 31.961 1322.603 28099.598 207.937025 1775 25.586 1321.685 2478338.5 18339.7049 1776 30.808 1321.321 58397.359 432.140457 1777 45.004 1321.021 66336.734 490.891832 1778 20.636 1320.662 25817.596 191.05021 1779 32.429 1319.441 11738.492 86.8648408 1780 31.4 1319.42 6886.302 50.9586348 1781 52.954 1319.408 3111.002 23.0214148 1782 50.24 1319.394 4092.932 30.2876968 1783 48.515 1319.389 12137.362 89.8164788 1784 34.136 1319.387 43983.848 325.480475 1785 35.227 1319.362 4880.322 36.1143828 1786 45 1318.861 87233.422 645.527323 1787 31.907 1318.611 13398.739 99.1506686 1788 41.768 1318.589 232563.891 1720.97279 1789 39.529 1318.407 9566.113 70.7892362 1790 46.954 1318.405 13212.546 97.7728404 1791 53.154 1318.387 4069.485 30.114189 1792 39.074 1318.375 7447.503 55.1115222 1793 59.704 1318.363 36163.148 267.607295 1794 30.603 1314.452 239448.859 1771.92156 1795 21.917 1312.679 77039.18 570.089932 1796 26.005 1312.635 141686.484 1048.47998 1797 52.047 1311.498 829.617 6.1391658 1798 25.103 1310.563 660235.938 4885.74594 1799 60.227 1306.401 3903.396 28.8851304 1800 45.07 1303.966 34127.25 252.54165 1801 25.985 1302.62 19980.832 147.858157 1802 51.644 1302.438 2354.193 17.4210282 1803 59.746 1302.393 45806.871 338.970845 1804 22.252 1301.272 57392.363 424.703486 1805 54.153 1300.353 8612.625 63.733425 1806 21.95 1299.873 83524.633 618.082284 1807 51.573 1299.424 30573.758 226.245809 1808 53.107 1299.416 25302.227 187.23648 1809 19.258 1298.642 21156.547 156.558448 1810 59.374 1298.359 12482.001 92.3668074 1811 45.03 1297.919 185944.781 1375.99138 1812 19.022 1296.685 28328.535 209.631159 1813 59.676 1295.779 9187.956 67.9908744 1814 28.449 1295.485 170789.578 1263.84288 1815 25.008 1294.604 46179.98 341.731852 1816 30.249 1293.53 103213.75 763.78175 1817 23.194 1292.502 19937.287 147.535924 1818 30.646 1290.433 8808.077 65.1797698 1819 28.43 1289.456 18305.74 135.462476 1820 58.946 1289.403 1384.637 10.2463138 1821 59.627 1289.387 2301.153 17.0285322 1822 19.693 1289.386 4377.44 32.393056 1823 25.244 1288.534 2999592.25 22196.9827 1824 59.353 1286.338 15882.02 117.526948 1825 30.82 1283.472 39585.328 292.931427 1826 51.523 1283.445 6039.294 44.6907756 1827 53.186 1283.43 8806.266 65.1663684 1828 34.802 1283.335 8705.754 64.4225796 1829 59.03 1282.394 15341.116 113.524258 1830 55.529 1282.378 7111.861 52.6277714 1831 45.022 1280.883 30969.486 229.174196 1832 19.518 1280.665 11716.79 86.704246 1833 59.668 1280.411 49052 362.9848 1834 28.452 1279.513 51832.395 383.559723 1835 51.44 1277.454 82467.625 610.260425 1836 52.927 1277.446 43342.348 320.733375 1837 45.003 1276.982 70758.93 523.616082 1838 30.44 1276.942 232610.75 1721.31955 1839 50.608 1276.907 1109.392 8.2095008 1840 23.116 1276.515 122081.953 903.406452 1841 44.995 1274.825 81134.063 600.392066 1842 26.295 1272.61 41668.891 308.349793 1843 25.135 1272.604 1010380.88 7476.81848 1844 20.731 1272.48 34951.859 258.643757 1845 30.456 1272.332 289698.906 2143.7719 1846 31.95 1270.569 58033.617 429.448766 1847 54.813 1270.498 1479.774 10.9503276 1848 59.96 1270.475 3445.866 25.4994084 1849 57.989 1270.447 1281.669 9.4843506 1850 59.188 1270.424 5638.458 41.7245892 1851 37.091 1269.475 1353.805 10.018157 1852 40.97 1269.437 2427.428 17.9629672 1853 35.834 1269.418 2728.887 20.1937638 1854 40.087 1269.413 1651.728 12.2227872 1855 39.127 1269.409 5886.622 43.5610028 1856 36.13 1269.399 4146.72 30.685728 1857 30.571 1267.515 21216.277 157.00045 1858 59.349 1266.399 6219.467 46.0240558 1859 54.894 1266.362 4477.317 33.1321458 1860 22.184 1263.606 94234.227 697.33328 1861 59.935 1263.437 57252.406 423.667804 1862 34.938 1263.312 16850.229 124.691695 1863 37.489 1262.942 10585.427 78.3321598 1864 55.342 1262.696 14468.394 107.066116 1865 52.525 1262.35 8346.335 61.762879 1866 39.287 1262.338 12846.079 95.0609846 1867 40.214 1262.335 8975.618 66.4195732 1868 58.04 1262.315 3522.354 26.0654196 1869 21.765 1262.289 123903.367 916.884916 1870 51.525 1261.458 99836.336 738.788886 1871 53.043 1261.455 83416.992 617.285741 1872 23.116 1260.537 46480.016 343.952118 1873 34.799 1259.311 14362.161 106.279991 1874 28.459 1257.534 298317.219 2207.54742 1875 31.559 1255.699 116446.039 861.700689 1876 30.3 1255.583 846248.25 6262.23705 1877 32.837 1255.567 35492.637 262.645514 1878 33.291 1255.561 28837.1 213.39454 1879 51.244 1255.489 2861.904 21.1780896 1880 32.024 1254.973 95395.18 705.924332 1881 23.278 1254.568 121895.391 902.025893 1882 59.74 1254.366 5949.095 44.023303 1883 57.787 1254.364 4684.266 34.6635684 1884 58.949 1254.347 2524.573 18.6818402 1885 57.081 1254.319 8885.642 65.7537508 1886 55.421 1254.301 4541.622 33.6080028 1887 45.012 1253.896 147699.641 1092.97734 1888 28.407 1251.496 141158.859 1044.57556 1889 25.249 1250.631 3974873.75 34371.53 29414.0658 1890 58.643 1250.455 11072.911 81.9395414 1891 18.98 1249.756 27491.875 203.439875 1892 31.656 1248.538 26623.307 197.012472 1893 32.92 1247.361 41003.309 303.424487 1894 51.498 1245.498 8761.765 64.837061 1895 53.085 1245.476 44366.938 328.315341 1896 19.632 1244.64 30831.113 228.150236 1897 59.714 1242.427 56404.984 417.396882 1898 56.779 1241.078 2758.177 20.4105098 1899 30.278 1239.607 317820.063 2351.86847 1900 31.547 1239.563 59098.148 437.326295 1901 28.472 1239.526 69276.641 512.647143 1902 51.321 1239.492 69059.977 511.04383 1903 52.866 1239.474 32897.426 243.440952 1904 60.102 1238.604 6424.091 47.5382734 1905 23.198 1238.572 492393.188 3643.70959 1906 25.057 1238.532 9391.734 69.4988316 1907 37.49 1237.332 6531.968 48.3365632 1908 45.022 1236.87 46682.961 345.453911 1909 19.717 1236.611 7412.524 54.8526776 1910 40.236 1236.283 8209.991 60.7539334 1911 52.172 1235.898 888.905 6.577897 1912 19.301 1235.868 4725.906 34.9717044 1913 30.417 1234.37 856949.25 6341.42445 1914 31.611 1233.532 122064.055 903.274007 1915 25.133 1232.595 24227.906 179.286504 1916 21.214 1231.482 60734.938 449.438541 1917 32.839 1231.084 60950.719 451.035321 1918 42.855 1230.887 154045.125 1139.93393 1919 30.565 1229.563 91573.625 677.644825 1920 22.209 1228.627 18700.881 138.386519 1921 60.379 1227.376 3367.176 24.9171024 1922 52.522 1227.324 6150.93 45.516882 1923 51.79 1227.323 4718.815 34.919231 1924 49.813 1227.305 11513.576 85.2004624 1925 40.192 1227.301 7194.075 53.236155 1926 59.636 1226.901 24848.395 183.878123 1927 21.859 1224.549 44181.957 326.946482 1928 30.406 1224.415 69779.727 516.36998 1929 23.094 1222.581 91712.016 678.668918 1930 51.595 1222.471 3662.568 27.1030032 1931 30.29 1218.52 476128.844 3523.35345 1932 31.528 1217.669 163110.922 1207.02082 1933 25.119 1217.539 169168.734 1251.84863 1934 23.259 1216.597 263113.625 1947.04083 1935 44.996 1215.355 16854.1 124.72034 1936 31.638 1211.447 117543.125 869.819125 1937 55.57 1210.742 2894.646 21.4203804 1938 20.68 1210.615 33522.359 248.065457 1939 31.023 1210.478 47086.57 348.440618 1940 60.002 1210.378 2460.045 18.204333 1941 59.452 1210.36 2822.82 20.888868 1942 53.367 1210.321 12792.264 94.6627536 1943 56.782 1210.2 4122.384 30.5056416 1944 45.004 1209.865 121766.766 901.074068 1945 25.804 1209.47 31311.213 231.702976 1946 20.729 1206.596 10410.285 77.036109 1947 51.214 1205.491 3944.53 29.189522 1948 31.553 1203.616 23597.301 174.620027 1949 49.516 1203.133 8821.522 65.2792628 1950 30.195 1202.589 39710.629 293.858655 1951 50.689 1202.365 25647.943 189.794778 1952 51.135 1201.518 6787.769 50.2294906 1953 42.67 1200.783 72117.422 533.668923 1954 23.167 1200.609 1313535.88 9720.16548 1955 25.206 1200.547 16658.26 123.271124 1956 46.71 1200.503 19274.873 142.63406 1957 30.044 1198.562 22444.525 166.089485 1958 59.87 1198.361 4803.752 35.5477648 1959 59.432 1198.353 4145.694 30.6781356 1960 19.706 1198.222 12631.365 93.472101 1961 21.013 1197.031 87272.023 645.81297 1962 30.406 1196.417 620780.5 4593.7757 1963 60.6 1196.352 7650.589 56.6143586 1964 31.578 1195.579 464075.188 3434.15639 1965 59.222 1190.363 5847.735 43.273239 1966 53.252 1188.334 55063.84 407.472416 1967 59.825 1187.418 3255.71 24.092254 1968 21.882 1186.597 192521.25 1424.65725 1969 60.255 1186.382 6004.868 44.4360232 1970 30.174 1185.657 58927.867 436.066216 1971 42.453 1184.524 53516.508 396.022159 1972 59.174 1184.402 4914.623 36.3682102 1973 21.325 1183.635 234638.141 1736.32224 1974 59.7 1183.443 7250.531 53.6539294 1975 31.801 1182.586 11399.009 84.3526666 1976 31.299 1181.569 94998.031 702.985429 1977 49.745 1181.254 3827.925 28.326645 1978 56.778 1181.221 3641.417 26.9464858 1979 60.62 1180.368 5690.042 42.1063108 1980 50.733 1180.335 38225.785 282.870809 1981 31.583 1179.597 236420.203 1749.5095 1982 19.143 1178.579 11057.592 81.8261808 1983 20.779 1178.495 131686.141 974.477443 1984 51.247 1178.431 6476.849 47.9286826 1985 41.654 1178.397 6623.212 49.0117688 1986 56.782 1178.216 4255.032 31.4872368 1987 25.333 1175.511 22551.197 166.878858 1988 24.101 1172.463 51734.363 382.834286 1989 53.214 1172.348 38737.391 286.656693 1990 44.949 1170.829 10637.769 78.7194906 1991 22.67 1169.26 18541.186 137.204776 1992 54.977 1169.167 7155.165 52.948221 1993 60.253 1166.411 2237.358 16.5564492 1994 53.22 1166.339 140311.391 1038.30429 1995 31.322 1165.301 58614.336 433.746086 1996 31.542 1163.587 34373.684 254.365262 1997 42.864 1163.561 317640.313 2350.53832 1998 20.221 1162.602 72301.289 535.029539 1999 30.294 1160.457 18931.172 140.090673 2000 20.533 1159.644 11926.854 88.2587196 2001 25.361 1158.633 29713.969 219.883371 2002 31.529 1157.615 44626.973 330.2396 2003 24.123 1156.492 33221.211 245.836961 2004 19.431 1154.638 47105.656 348.581854 2005 31.311 1154.523 72400.438 535.763241 2006 23.924 1152.527 201380.953 1490.21905 2007 30.384 1152.393 58578.801 433.483127 2008 21.985 1150.625 37468.707 277.268432 2009 53.194 1150.361 120706.516 893.228218 2010 30.027 1149.312 119640.453 885.339352 2011 42.541 1147.575 30441.695 225.268543 2012 23.629 1144.649 29225.717 216.270306 2013 31.311 1143.136 23237.896 171.96043 2014 24.001 1142.545 23198.781 171.670979 2015 50.597 1142.373 118697.242 878.359591 2016 23.275 1141.59 47004.074 347.830148 2017 21.014 1140.488 174479.281 1291.14668 2018 41.649 1140.415 5001.429 37.0105746 2019 19.442 1138.647 71437.797 528.639698 2020 31.367 1138.553 111894.984 828.022882 2021 25.359 1137.454 68654.234 508.041332 2022 53.625 1137.364 4344.629 32.1502546 2023 23.802 1136.534 21588.393 159.754108 2024 25.107 1135.542 37640.664 278.540914 2025 22.904 1134.648 71461.75 528.81695 2026 24.113 1134.504 347155.5 2568.9507 2027 60.091 1134.459 5499.415 40.695671 2028 59.353 1134.437 3032.865 22.443201 2029 53.188 1134.391 56996.746 421.77592 2030 51.575 1134.21 9499.726 70.2979724 2031 29.971 1133.34 41626.363 308.035086 2032 20.936 1128.605 47002.93 347.821682 2033 28.199 1128.485 105328.227 779.42888 2034 53.209 1128.38 354067.813 2620.10182 2035 50.589 1126.389 28851.957 213.504482 2036 31.552 1125.231 11812.239 87.4105686 2037 41.868 1125.221 15596.393 115.413308 2038 32.903 1125.218 126022.156 932.563954 2039 31.908 1125.217 25750.453 190.553352 2040 39.864 1125.21 6728.264 49.7891536 2041 36.571 1125.206 22293.379 164.971005 2042 39.556 1125.202 16396.361 121.333071 2043 26.016 1125.161 31188 230.7912 2044 29.167 1125.148 41243.914 305.204964 2045 38.059 1124.829 29475.412 218.118049 2046 31.341 1122.552 34561.762 255.757039 2047 23.72 1122.456 36783.59 272.198566 2048 44.964 1121.808 44746.047 331.120748 2049 18.812 1119.758 5198.635 38.469899 2050 24.134 1118.525 119093.102 881.288955 2051 25.151 1118.499 30842.926 228.237652 2052 23.899 1114.554 208412.047 1542.24915 2053 29.989 1111.346 183555.891 1358.31359 2054 28.181 1110.479 70840.586 524.220336 2055 51.147 1110.414 125425.094 928.145696 2056 20.151 1108.578 25817.43 191.048982 2057 23.801 1106.903 187048.984 1384.16248 2058 50.557 1104.421 171816.922 1271.44522 2059 58.615 1102.375 6383.561 47.2383514 2060 51.593 1101.214 1985.06 14.689444 2061 49.777 1100.828 2256.389 16.6972786 2062 19.219 1100.543 6005.286 44.4391164 2063 21.831 1100.522 38794.496 287.07927 2064 25.329 1099.552 13957.11 103.282614 2065 53.545 1099.417 30059.629 222.441255 2066 21.401 1098.745 112194.32 830.237968 2067 52.17 1098.418 3632.612 26.8813288 2068 20.123 1097.551 4589.071 33.9591254 2069 24.128 1096.552 522675.844 3867.80125 2070 53.285 1096.434 26559.404 196.53959 2071 51.387 1096.263 109630.906 811.268704 2072 29.987 1095.392 58529 433.1146 2073 19.018 1094.643 6701.293 49.5895682 2074 20.501 1091.546 15707.116 116.232658 2075 23.598 1090.488 10901.328 80.6698272 2076 53.19 1090.433 338706.531 2506.42833 2077 20.982 1082.62 65489.449 484.621923 2078 21.397 1082.584 32035.098 237.059725 2079 19.289 1080.554 12653.619 93.6367806 2080 53.408 1076.445 4457.142 32.9828508 2081 53.657 1076.436 3256.97 24.101578 2082 52.905 1076.431 2378.771 17.6029054 2083 20.569 1075.555 86250.758 638.255609 2084 29.991 1073.413 40851.953 302.304452 2085 23.494 1068.813 147886.688 1094.36149 2086 21.443 1068.575 62297.086 460.998436 2087 18.988 1068.472 18219.826 134.826712 2088 23.8 1064.568 7053.475 52.195715 2089 20.279 1063.503 21330.266 157.843968 2090 19.116 1062.663 4397.519 32.5416406 2091 32.024 1060.467 45340.27 335.517998 2092 60.603 1060.416 6794.908 50.2823192 2093 52.255 1058.393 12166.359 90.0310566 2094 54.069 1058.366 9593.294 70.9903756 2095 49.741 1057.321 5347.421 39.5709154 2096 23.962 1054.469 82210.703 608.359202 2097 23.588 1051.388 89102.742 659.360291 2098 30.345 1044.353 4195.99 31.050326 2099 23.401 1040.537 10620.867 78.5944158 2100 20.577 1032.597 21339.391 157.911493 2101 24.092 1025.286 62953.594 465.856596 2102 19.87 1018.527 7472.115 55.293651 2103 23.953 1016.515 121646.805 900.186357 2104 30.418 1016.354 134689.156 996.699754 2105 20.528 1015.594 21665.502 160.324715 2106 23.573 1013.438 115542.805 855.016757 2107 30.392 1011.276 33455.215 247.568591 2108 18.922 1010.399 9148.562 67.6993588 2109 20.423 1006.679 8503.45 62.92553 2110 47.1 998.211 46778.898 346.163845 2111 23.462 995.402 32905.055 243.497407 2112 51.388 991.214 2437.004 18.0338296 2113 21.942 988.585 32196.633 238.255084 2114 20.878 988.565 12280.876 90.8784824 2115 29.367 987.262 48744.594 360.709996 2116 23.484 984.509 27134.879 200.798105 2117 47.966 984.342 3780.828 27.9781272 2118 23.398 981.665 20057.672 148.426773 2119 30.895 981.502 25569.025 189.210785 2120 19.773 978.532 4305.65 31.86181 2121 30.386 973.323 32013.334 236.898672 2122 29.291 971.253 5491.579 40.6376846 2123 20.901 969.104 41305.473 305.6605 2124 25.141 965.491 48757.68 360.806832 2125 24.072 964.44 7556.274 55.9164276 2126 20.829 963.206 16317.524 120.749678 2127 28.81 962.49 19231.721 142.314735 2128 20.219 960.462 9783.454 72.3975596 2129 23.379 956.597 24888.623 184.17581 2130 22.899 956.484 12570.926 93.0248524 2131 59.245 955.564 1391.343 10.2959382 2132 19.095 954.938 8325.797 61.6108978 2133 20.123 952.494 5814.21 43.025154 2134 60.591 950.571 4099.453 30.3359522 2135 40.291 950.357 19953.055 147.652607 2136 41.194 950.357 47130.363 348.764686 2137 24.004 949.406 63025.402 466.387975 2138 29.386 949.317 56691.895 419.520023 2139 51.536 948.37 24475.225 181.116665 2140 60.327 947.639 4107.147 30.3928878 2141 23.951 945.481 43559.406 322.339604 2142 20.894 944.588 148397.719 1098.14312 2143 30.047 944.458 13261.684 98.1364616 2144 23.976 933.488 10559.713 78.1418762 2145 20.085 931.546 45600.914 337.446764 2146 18.961 926.553 4596.708 34.0156392 2147 20.898 925.264 49014.879 362.710105 2148 20.978 920.557 5005.199 37.0384726 2149 18.938 918.591 4865.919 36.0078006 2150 56.883 918.079 1112.862 8.2351788 2151 51.665 915.677 1583.223 11.7158502 2152 52.923 915.395 2432.005 17.996837 2153 29.904 914.917 25552.555 189.088907 2154 40.82 912.413 454771.625 3365.31003 2155 23.975 911.5 11979.838 88.6508012 2156 18.986 911.287 4659.146 34.4776804 2157 29.339 906.271 22190.25 164.20785 2158 51.09 902.979 700.182 5.1813468 2159 20.832 902.486 52433.75 388.00975 2160 49.645 900.36 24044.1 177.92634 2161 24.096 898.383 45429.156 336.175754 2162 56.504 897.659 5498.265 40.687161 2163 56.278 896.829 1467.63 10.860462 2164 22.577 896.41 21323.018 157.790333 2165 19.262 895.584 4205.946 31.1240004 2166 23.992 882.405 17175.494 127.098656 2167 55.786 880.774 2373.386 17.5630564 2168 59.474 879.635 1745.675 12.917995 2169 19.949 879.567 5377.363 39.7924862 2170 29.754 876.473 23381.605 173.023877 2171 42.753 875.379 114573.516 847.844018 2172 21.028 872.459 5747.54 42.531796 2173 19.956 868.588 5911.415 43.744471 2174 51.593 867.774 1445.617 10.6975658 2175 55.321 867.675 724.974 5.3648076 2176 59.198 867.635 1190.664 8.8109136 2177 58.799 867.622 2502.561 18.5189514 2178 20.829 852.468 7367.667 54.5207358 2179 49.047 846.946 1713.276 12.6782424 2180 57.438 846.946 2112.215 15.630391 2181 60.566 846.924 2585.42 19.132108 2182 22.644 846.426 1215.356 8.9936344 2183 29.086 841.303 8721.339 64.5379086 2184 53.187 837.301 11143.638 82.4629212 2185 55.22 835.737 611.264 4.5233536 2186 54.381 832.253 1154.893 8.5462082 2187 59.347 824.672 836.045 6.186733 2188 20.776 824.421 2193.866 16.2346084 2189 59.758 824.025 5333.992 39.4715408 2190 58.792 823.646 7006.314 51.8467236 2191 53.249 821.319 3106.751 22.9899574 2192 60.055 815.685 1288.741 9.5366834 2193 29.084 815.416 2310.018 17.0941332 2194 50.654 813.317 13369.253 98.9324722 2195 53.192 799.334 37386.438 276.659641

The quality of the regression is shown in FIG. 2. 

1. A process for normalizing the concentration of at least one analyte in a urine sample, comprising the following steps: a) determining the concentration of at least one analyte; b) determining the concentration of at least one collagen or collagen fragment; c) normalizing the concentration of said at least one analyte relative to the concentration of said at least one collagen or collagen fragment.
 2. The process according to claim 1, wherein the urine sample is subdivided into several subsamples before the concentration of the analyte is determined.
 3. The process according to claim 2, wherein said separation is effected by: chromatographic methods, such as affinity chromatography, ion-exchange chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, gel filtration, optionally using FPLC or HPLC; adsorption to specific materials, such as SELDI process, ClinProt process; electrophoretic methods, such as gel electrophoresis or capillary electrophoresis.
 4. The process according to claim 1, wherein said determination of the contents of analytes and/or collagen or collagen fragments in said subsamples is effected by a UV/Vis detector, fluorescence detector, refractive index detector, diode array detector, mass spectrometer, gel scanner, staining or derivatization, followed by detection with suitable means.
 5. The process according to claim 1, wherein said at least one collagen or collagen fragment is identified by its migration or retention time and optionally further suitable parameters, such as mass spectra, UV/Vis spectra, fluorescence spectra, affinity for particular ligands, for example, antibodies.
 6. The process according to claim 1, wherein said at least one collagen fragment is selected from the peptides of SEQ ID Nos. 1 to
 42. 7. The process according to claim 1, wherein the concentrations of at least two or at least three, preferably at least 5, 10, 20, 30, 40 or 50 collagens or collagen fragments are determined and employed for normalization.
 8. The process according to claim 1, wherein said normalization is effected by linear regression.
 9. Use of collagens or collagen fragments for the normalization of analyte concentrations in urine samples.
 10. The use according to claim 9, wherein said collagen fragments are selected from the peptides of SEQ ID Nos. 1 to
 42. 11. The use according to claim 9, wherein at least two or at least three, preferably at least 5, 10, 20, 30, 40 or 50 collagens or collagen fragments are employed for normalization. 